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  Reagent for determining laminin 5 antigen in biological sample and assay methodUSPTO Application #: 20070269835Title: Reagent for determining laminin 5 antigen in biological sample and assay method Abstract: A method of determining a laminin 5 antigen in a biological sample, comprising the steps of bringing an antibody reactive to a laminin 5 γ2 chain N-terminal fragment into contact with the biological sample; measuring a reaction of the antibody; and determining an amount of the laminin 5 antigen based on a measurement result of the reaction, as well as, a method of detecting a laminin 5-producing tumor cell, a method of examining acute respiratory distress syndrome and a method of evaluating malignancy of a malignant tumor based on the assay method. (end of abstract) Agent: Choate, Hall & Stewart LLP - Boston, MA, US Inventors: Masahiko Katayama, Noriko Sanzen, Kiyotoshi Sekiguchi USPTO Applicaton #: 20070269835 - Class: 435007200 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Involving A Micro-organism Or Cell Membrane Bound Antigen Or Cell Membrane Bound Receptor Or Cell Membrane Bound Antibody Or Microbial Lysate The Patent Description & Claims data below is from USPTO Patent Application 20070269835. Brief Patent Description - Full Patent Description - Patent Application Claims
TECHNICAL FIELD [0001] The present invention relates to a method and a reagent for determining a laminin 5 antigen in a biological sample. BACKGROUND ART [0002] A basal lamina is constituted by an extracellular matrix component which is mainly a collagen, and exists universally in an organism. One of macromolecular proteins constituting the basal lamina is laminin (hereinafter, often abbreviated as LN). The LN is classified into more than ten kinds based on the structure thereof, which are different in functions and localized tissues, and is classified by adding a number at the end, e.g., LN1 or LN2. Every LN is constituted by a complex formed from three polypeptide chains each having a different amino acid sequence, and gives a cross-like molecular form in an electron microscope observation. The three polypeptide chains are respectively called .alpha. chain, .beta. chain, and .gamma. chain, and each has several molecular species (.alpha.1 to .alpha.5, .beta.1 to .beta.3, and .gamma.1 and .gamma.2). [0003] In the basal lamia between epithelial cells and a connective tissue backing the epithelial cells, there exists a cell adhesion structure peculiar to the epithelial cells. As an extracellular matrix-constituting protein which exists mainly in the above structure, laminin 5 is known (hereinafter, often abbreviated as LN5). Physiological characteristics of LN5 is that it is the only LN in the above LN group, produced solely from epithelial cells and that it has an activity to promote adhesion to the basal lamia and motility of epithelial cells. As for the epithelial cell, it is known phenomena that it adheres strongly to LN5 and the basal lamia containing LN5 via a specific receptor called integrin on a cell membrane of the epithelial cell itself, and that it migrates aggressively. [0004] The LN5 is constituted by a complex formed from one .alpha.3 chain, one .beta.3 chain, and one .gamma.2 chain. The .gamma.2 chain, in particular, is considered to be specific to LN5, and is not included in the other LN molecular species [Dev. Dyn., 218, 213-234 (2000)]. In addition, it is found that LN5 is partially degraded by a proteolytic enzyme (protease) when secreted from the epithelial cells, and it is indicated that, especially by shedding an N-terminal part of the .gamma.2 chain, the remaining molecular entity of LN5 from which the N-terminal part was released has an increased activity for promoting cell movement [J. Cell Biol., 148, 615-624 (2000)]. [0005] That is, the amount of LN5 .gamma.2 chain fragments released from the epithelial cells, etc. reflects an LN5 production amount in the cells, and it considered to be a potential index for measuring an epithelial cell motility-promoting activity of LN5 in an epithelial tissue. [0006] Recently, a lot of study results reported that an expression of the LN5 increased in a malignant tumor tissue from epithelial cells. There are many reports, in particular, that the LN5 expression level correlates well with an invasiveness of a malignant tumor, and its application as a pathological marker for the purpose of in vitro diagnosis of cancer is considered to be possible [J. Natl. Cancer Inst., 91, 1882-1887 (1999) and Cancer, 85, 2315-2321 (1999)]. [0007] However, in the above studies, there is mainly employed a method in that a pathologic tissue is removed from a body of a patient through an operation to prepare a section, and then an expression site of a target protein is immunstained with an antibody. Such method lacks quantitativeness and versatility in terms of practical application to in vitro diagnosis in medical places, and also has a problem of imposing physical strains on a patient, for example, a surgical operation and a tissue biopsy. Therefore, an establishment of a simple and quick method of determining a level of an LN5 antigen in a biological sample, such as blood, which can be sampled relatively safely is desired. [0008] Recently, there has been reported an enzyme-linked immunosorbent assay (ELISA) using two kinds of monoclonal antibodies to LN5 (a monoclonal antibody BM165 against an .alpha.3 chain and a monoclonal antibody 6F12 against a .beta.3 chain) [J. Immunol. Meth., 224, 161-169 (1999)]. However, this report shows no example that an LN5 antigen in a biological sample is detected by ELISA. DISCLOSURE OF THE INVENTION [0009] An object of the present invention is to provide a method of determining an LN5 antigen in a small amount of a biological sample accurately and simply, using an antibody that specifically binds to LN5. [0010] The inventors of the present invention focused on the degradation of an LN5.gamma.2 chain in various examinations of a method of determining an LN5 antigen in a biological sample. Thus, the inventors of the present invention prepared monoclonal antibodies recognizing respective chains of LN5, and immunologically analyzed an LN5 antigen in culture supernatants of epithelial tumor cells. As a result, it was found that many epithelial tumor cells secreted LN5 with its .gamma.2 chain shed into culture supernatants thereof. Furthermore, it was confirmed that the prepared monoclonal antibody against the .gamma.2 chain was reactive to a .gamma.2 chain fragment released due to the protease degradation of LN5. [0011] As a result of further analyses, a plurality of kinds of monoclonal antibodies reactive to the released .gamma.2 chain fragment were prepared, to prepare a sandwich-type immunological assay reagent using two different monoclonal antibodies reactive to the .gamma.2 chain fragment. It was also found that an LN5 antigen in a blood serum and a blood plasma could be determined efficiently by using the assay reagent using two different monoclonal antibodies reactive to the LN5 .gamma.2 chain fragment. [0012] As explained above, the .gamma.2 chain is a constituent specific to LN5, and not included in the other LN molecular species. That is, the assay reagent using the antibody against the .gamma.2 chain fragment allows to determine any molecule of a .gamma.2 chain fragment, a nonfragmented .gamma.2 chain, a .beta.3 chain-.gamma.2 chain complex, or an .alpha.3 chain-.beta.3 chain-.gamma.2 chain complex in a biological sample. It is therefore considered that the reagent determines most of LN5 antigens and thus may provide a useful index which reflects the LN5-producing amount in an epithelial tissue for grasping a pathological condition. [0013] It was also experimentally examined what kind of in vivo phenomena an increase of an LN5 antigen in a biological sample reflects. Eleven kinds of human pancreatic tumor cell lines were cultured in a liquid medium in the presence of a 10% bovine fetal serum for a given period, and LN5 concentrations in the culture supernatants and expression levels of various integrins which are adhesion molecular receptors expressed were compared. Six kinds of LN5-producing lines obviously had a tendency to express higher expression level of .beta.4 integrin in comparison to five kinds of LN5-nonproducing lines. That is, it was experimentally proved that the in vivo LN5 antigen-producing amount may be an index for the .beta.4 integrin expression level in epithelial cells in an in vivo epithelial tissue. [0014] Further, a human tumor producing LN5 was transplanted to a nude mouse and it was thus found that a growth of the tumor and the LN5 concentration in a blood serum correlated each other, thereby elucidating that determining LN5 in a blood serum allows monitoring of the growth of an LN5-producing tumor. [0015] In addition, it was hypothesized that an LN5 metabolism in an organism increases in pulmonary inflammatory disease, based on the fact that much LN5 is included especially in a pulmonary epithelial tissue among organs in an organism. Under the hypothesis, the LN5 antigen concentration in blood plasma specimens sampled from patients suffering from acute respiratory distress syndrome (hereinafter, abbreviated as ARDS) and in blood plasma specimens sampled from healthy subjects was determined in accordance with the above method. It was found that the concentration in blood was obviously higher for a group of the patients suffering from acute respiratory distress syndrome, in comparison with the group of healthy subjects, thereby discovering that the method was extremely useful for clinical diagnosis of the disease. [0016] The present invention was completed based on the above findings. That is, the present invention provides the following. [0017] 1. A method of determining a laminin 5 antigen in a biological sample, comprising the steps of: [0018] bringing an antibody reactive to a laminin 5 .gamma.2 chain N-terminal fragment into contact with the biological sample; [0019] measuring a reaction of the antibody; and [0020] determining an amount of the laminin 5 antigen based on a measurement result of the reaction. [0021] 2. A method according to item 1, wherein the antibody is a monoclonal antibody produced from a cell deposited with an accession number of FERM BP-8136, FERM BP-8133, or FERM BP-8134. Continue reading... Full patent description for Reagent for determining laminin 5 antigen in biological sample and assay method Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Reagent for determining laminin 5 antigen in biological sample and assay method patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. 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